sounds interesting. I haven´t heard of that phenomenon either, but I
wouldn´t be too surprised if PNK had a nucleotide preference. As you
seem to have done quite some controls (and literature research) on that,
PUBLISH IT! I personally would try Anal.Biochem. BioTechniques, or even
NAR.
Can you also see the effect with natural DNA fragments? An experiment
could be to digest a common vector (pUC, pBluescript...) with different
restriction enzymes so to leave different terminal residues (e.g. EcoRI
for A, BamHI for G, XhoI for T, XbaI for C; all these make a 4-base 5´
extension that should label well with PNK). Then use PNK to label the
linearized plasmid, and determine the efficiency. What you would see
then is the exchange reaction of PNK (is it also dependent on the
terminal nt?). In a control, dephosphorylate the DNAs by alkaline
phosphatase (shrimps is good in my hands), then repeat the kinase
reaction. That should give you the base preference seen with oligos. If
it doesn´t, your could heat-denature the plasmid (base-preference only
with single strands?) and repeat the labelling. If you cannot reproduce
the effect with natural DNA fragments, I´d suspect the primer synthesis
is causing a problem (probably residual ammonium ions which strongly
inhibit PNK?)
Frank
PS: Would you mind to let us know which nucleotide is best....?
January Weiner wrote:
>> Hello,
> I had to label about 40-50 primers, and while doing this I have spotted a
> strange phenomenon: the labelling efficiency could drop to less then 10%
> depending on the first nucleotide of the primer, independently of the
> sequence and condition. Maybe I am just illiterate and don't know some
> basic facts about PNK, but I could not find any literature reporting this
> phenomenon. It is reproducible and independent from the company which
> synthetized oligos. Any clues?
>> Cheers,
> j.
>> --
> ----)-\//-///-----------------------------------January-Weiner-3-------
> Sacred cows make the best hamburger. - Mark Twain